Recessive mutations in the ubiquitously expressed POLR3A gene cause one of the most frequent forms of childhood-onset hypomyelinating leukodystrophy (HLD): POLR3-HLD. POLR3A encodes the largest subunit of RNA Polymerase III (Pol III), which is responsible for the transcription of transfer RNAs (tRNAs) and a large array of other small non-coding RNAs. In order to study the central nervous system pathophysiology of the disease, we introduced the French Canadian founder Polr3a mutation c.2015G&thinsp;&gt;&thinsp;A (p.G672E) in mice, generating homozygous knock-in (KI/KI) as well as compound heterozygous mice for one Polr3a KI and one allele (KI/KO). Both KI/KI and KI/KO mice are viable and are able to reproduce. To establish if they manifest a motor phenotype, WT, KI/KI and KI/KO mice were submitted to a battery of behavioral tests over one year. The KI/KI and KI/KO mice have overall normal balance, muscle strength and general locomotion. Cerebral and cerebellar Luxol Fast Blue staining and measurement of levels of myelin proteins showed no significant differences between the three groups, suggesting that myelination is not overtly impaired in Polr3a KI/KI and KI/KO mice. Finally, expression levels of several Pol III transcripts in the brain showed no statistically significant differences. We conclude that the first transgenic mice with a leukodystrophy-causing Polr3a mutation do not recapitulate the childhood-onset HLD observed in the majority of human patients with POLR3A mutations, and provide essential information to guide selection of Polr3a mutations for developing future mouse models of the disease.

Electronic supplementary material: The online version of this article (doi:10.1186/s13041-017-0294-y) contains supplementary material, which is available to authorized users.

Fig3: No Purkinje cell loss in Polr3a KI/KI and KI/KO mice. a) Nissl staining of sagittal cerebellar sections of 365 days old mice. Staining was performed on four mice per group and representative image are shown for each group. Scale bar = 100 μm (top) and 50 μm (bottom). b) Purkinje cell counts of mid-sagittal cerebellar sections of 365 days old mice (n = 4 per group). Data are represented as mean +/− SEM

Mentions:
Hypomyelination is the main pathological feature of POLR3-HLD [5, 33]. Thus, to assess whether Polr3a KI/KI and KI/KO mice display hypomyelination, we stained coronal brain sections from 90 and 365 days old mice with Luxol Fast Blue (LFB), which is commonly used to detect myelin in the CNS. We observed normal and complete myelination in the brain and cerebellum of KI/KI and KI/KO mice, where the staining was indistinguishable from age-matched WT mice (Fig. 2a, b and Additional file 1: Figure S5). In addition, we measured the levels of the major protein components of myelin by western blot in the cerebellum of 90-day-old mice. Protein levels of Myelin Basic Protein (MBP), Proteolipid Protein (PLP), Myelin-associated Glycoprotein (MAG) and 2′,3′-Cyclic Nucleotide 3′ Phosphodiesterase (CNP) were comparable between WT, KI/KI and KI/KO mice (Fig. 2c). These results suggest that Polr3a KI/KI and KI/KO mice undergo normal gross myelination and do not experience major demyelination at one year of age. Since cerebellar atrophy and Purkinje cell loss is a major feature in POLR3-HLD [5, 33], we then evaluated cerebellar morphology using Nissl staining followed by Purkinje cell counts in 365-day-old mice. Cerebellar morphology was overall normal (Fig. 3a) as were Purkinje cell numbers (Fig. 3b), implying that KI/KI and KI/KO mice do not present cerebellar atrophy.Fig. 2

Fig3: No Purkinje cell loss in Polr3a KI/KI and KI/KO mice. a) Nissl staining of sagittal cerebellar sections of 365 days old mice. Staining was performed on four mice per group and representative image are shown for each group. Scale bar = 100 μm (top) and 50 μm (bottom). b) Purkinje cell counts of mid-sagittal cerebellar sections of 365 days old mice (n = 4 per group). Data are represented as mean +/− SEM

Mentions:
Hypomyelination is the main pathological feature of POLR3-HLD [5, 33]. Thus, to assess whether Polr3a KI/KI and KI/KO mice display hypomyelination, we stained coronal brain sections from 90 and 365 days old mice with Luxol Fast Blue (LFB), which is commonly used to detect myelin in the CNS. We observed normal and complete myelination in the brain and cerebellum of KI/KI and KI/KO mice, where the staining was indistinguishable from age-matched WT mice (Fig. 2a, b and Additional file 1: Figure S5). In addition, we measured the levels of the major protein components of myelin by western blot in the cerebellum of 90-day-old mice. Protein levels of Myelin Basic Protein (MBP), Proteolipid Protein (PLP), Myelin-associated Glycoprotein (MAG) and 2′,3′-Cyclic Nucleotide 3′ Phosphodiesterase (CNP) were comparable between WT, KI/KI and KI/KO mice (Fig. 2c). These results suggest that Polr3a KI/KI and KI/KO mice undergo normal gross myelination and do not experience major demyelination at one year of age. Since cerebellar atrophy and Purkinje cell loss is a major feature in POLR3-HLD [5, 33], we then evaluated cerebellar morphology using Nissl staining followed by Purkinje cell counts in 365-day-old mice. Cerebellar morphology was overall normal (Fig. 3a) as were Purkinje cell numbers (Fig. 3b), implying that KI/KI and KI/KO mice do not present cerebellar atrophy.Fig. 2

Recessive mutations in the ubiquitously expressed POLR3A gene cause one of the most frequent forms of childhood-onset hypomyelinating leukodystrophy (HLD): POLR3-HLD. POLR3A encodes the largest subunit of RNA Polymerase III (Pol III), which is responsible for the transcription of transfer RNAs (tRNAs) and a large array of other small non-coding RNAs. In order to study the central nervous system pathophysiology of the disease, we introduced the French Canadian founder Polr3a mutation c.2015G&thinsp;&gt;&thinsp;A (p.G672E) in mice, generating homozygous knock-in (KI/KI) as well as compound heterozygous mice for one Polr3a KI and one allele (KI/KO). Both KI/KI and KI/KO mice are viable and are able to reproduce. To establish if they manifest a motor phenotype, WT, KI/KI and KI/KO mice were submitted to a battery of behavioral tests over one year. The KI/KI and KI/KO mice have overall normal balance, muscle strength and general locomotion. Cerebral and cerebellar Luxol Fast Blue staining and measurement of levels of myelin proteins showed no significant differences between the three groups, suggesting that myelination is not overtly impaired in Polr3a KI/KI and KI/KO mice. Finally, expression levels of several Pol III transcripts in the brain showed no statistically significant differences. We conclude that the first transgenic mice with a leukodystrophy-causing Polr3a mutation do not recapitulate the childhood-onset HLD observed in the majority of human patients with POLR3A mutations, and provide essential information to guide selection of Polr3a mutations for developing future mouse models of the disease.

Electronic supplementary material: The online version of this article (doi:10.1186/s13041-017-0294-y) contains supplementary material, which is available to authorized users.